Sulfonation is a fundamental process in the biotransformation of endobiotics as well as xenobiotics. Sulfonation is essential for normal growth and development and maintenance of the internal milieu. Sulfonated macromolecules such as glycosaminoglycans and proteoglycans are involved in cell surface and connective tissue structures. The sulfonation of tyrosine residues is a widespread post-translational modification of many secretory and membrane proteins. Sulfolipids such as sphingolipids and galactoglycerolipids are concentrated in the brain, peripheral nerves and reproductive tissues. Additionally, sulfoconjugation is important in the metabolism of low molecular weight compounds such as neurotransmitters, iodothyronines and steroid hormones. Sulfonation requires the universal sulfonate donor molecule, 3-phosphoadenosine 5-phosphosulfate (PAPS), a fact that establises PAPS as a strategic biological molecule and makes its availability of vital importance. The biosynthesis of PAPS requires two catalytic reactions which are carried out by a bifunctional protein (PAPS synthase). We have cloned PAPS synthase from human and guinea pig, determined chromosomal localization, and examined tissue-specific expression. We have performed structure/function studies including the cloning and expression of the active ATP sulfurylase and APS kinase domains. We have identified a nucleotide-binding motif (HxGH) in the ATP sulfurylase domain of PAPS synthase and by site-selected mutagenesis determined that this conserved motif is essential for the ATP sulfurylase half-reaction. We have demonstrated that purified guinea pig and human PAPS synthase form a phosphoenzyme intermediate during the APS kinase half-reaction. Furthermore, by site-selected mutagenesis we determined that aspartate residues in a conserved phosphoryl transfer motif (DxDxI/V)located in the APS kinase domain are essential for this reaction to occur. Finally, we have cloned, overexpressed, and purified a second human PAPS synthase (75% identical to PAPS synthase 1) and found that it is biochemically and kinetically distinct from hunman PAPS synthase 1. Our initial studies on the differential tissue expression of these two isoforms has revealed very provocative results, e.g. both isoforms are expressed in the lung with PAPS synthase 1 being dominant, whereas only PAPS synthase 2 is expressed in the liver. Steroid sulfotransferases play a fundamental role in specific physiological systems and disorders, e.g. hormone-dependent cancers of the prostate and breast, obesity and diabetes, menstrual cycle and reproduction, fetal lung maturation and the respiratory distress syndrome, anxiety, stress and seizure disorders. Sulfonation has a marked effect on the biological activity of steroids, regardless of whether the steroid is acting via a genomic or nongenomic mechanism. In the genomic action of steroids only the free or unconjugated steroid form is capable of binding to its cognate nuclear receptor. On the other hand, in the nongenomic action of steroids (action at extranuclear sites on membranes), the functionally significant form of the steroid can be sulfoconjugated. In either version of steroid action, a crucial function of steroid sulfotransferases is to modulate the availability of the biologically active form of the steroid. As with PAPS synthase, important advances were made in the area of structure/function relationships involving 3-hydroxysteroid sulfotransferases (HST). For instance, we identified by chimera formation and site-selected mutagenesis a key amino acid residue responsible for the remarkable stereoisomerism exhibited by the 3 alpha- and 3 beta-HST isoforms. Furthermore, we discovered during the purification of testosterone sulfotransferase activity that the sulfonation of testosterone, as well as the sulfoconjugation of the 17 beta-hydroxyl group of estradiol, was carried out by the 3 alpha- HST isoform, whereas the 3 beta-HST isoform was unable to perform this function. Only estrogen sulfotransferase (EST) will carry out sulfonation involving the 3-hydroxyl group of estradiol, and neither the 3 alpha- nor 3 beta-HST isoform will act on this group. Furthermore, EST will not sulfonate the 17 beta-hydroxyl group of estradiol (or testosterone).The 3 alpha-HST isoformhas been overexpressed andis currently undergoing purification for use in crystallography and resolution of its three-dimensional structure.